Our long-term goal is to understand the contribution to gene regulation made by nucleosome positioning along the 100A chromatin fiber. While the bulk of the DNA is packaged in a nucleosome array, an active or inducible promoter lies in a nucleosome-free region, a DNase I hypersensitive site (DH site). Genetic and biochemical analyses have shown that GAGA factor, a sequence-specific DNA binding protein, plays a key role in establishing the correct pattern of DH sites at the hsp26 gene of Drosophila melanogaster. GAGA factor is an abundant nuclear protein, involved in the expression of many constitutive, inducible, and patterned genes. Mutations in the gene encoding GAGA factor (Trl) indicate a role in maintaining the active state of homeotic genes and in counteracting the silencing observed in position effect variegation. We propose that GAGA factor acts by directing local formation of DH sites, creating specific gaps in the nucleosome array that facilitate local gene expression and impact higher order structure. However, the mechanism of such DH site formation is incompletely understood. Our first specific aim is to determine whether the presence of GAGA factor in the 5' regulatory region of hsp26 is sufficient to direct formation of a DH site, or whether this transition requires the presence of an adjacent TFIID complex. In vivo footprinting, and immunoprecipitation of DNA/protein complexes, will be used to assess the pattern of GAGA factor, TBP, and nucleosome disposition on promoters with altered regulatory sequences. Aim two is to establish an in vivo system to examine possible differences in the roles of the different GAGA factor isoforms, looking at hsp26 DH sites, Ubx regulation, and metaphase chromosome structure. Isoforms tagged with a specific epitope will be used to recover potential protein complexes by affinity chromatography. The third aim is to utilize an unbiased strategy to identify gene products that interact functionally with GAGA factor to create DH sites. A genetic screen will be performed, looking for dominant suppressors and enhancers of a reporter gene that is driven by an hsp26 promoter weakened by an altered GAGA factor binding site. Future efforts will focus on factors found in both the biochemical and genetic analyses to interact with GAGA factor to alter the nucleosome array. These investigations will generate a much clearer picture of how GAGA factor functions to direct formation of a DH site, providing a useful example of this critical step leading to regulated gene expression. Such knowledge is important for our understanding of human growth and development, including health problems such as cancer and aging.